Herpes simplex virus type-1 (HSV-1) with limited treatment options is a leading cause of infectious blindness and an important indication for corneal transplants in the US. The current treatment options include acyclovir and its derivatives, ganciclovir and foscarnet. All these drugs primarily act upon viral thymidine kinase to inhibit viral DNA replication and in essence have a similar mechanism of action. While these options are effective and show promise in reducing ocular HSV-1 infection including herpes stromal keratitis (HSK), emergence of drug resistance in the recent years has caused significant distress in the clinical care of ocular HSV-1 patients. As a result, there exists an unmet need for the development of new treatments against HSV-1 especially the ones that rely on novel modes of action. In our previous 3-year funding period we have characterized two candidates that target two different stages of HSV-1 infection; viral entry and viral protein synthesis. Against viral entry a highly effective anti-HSV-1 aptamer (DApt) was developed that targets HSV-1 glycoprotein D (gD). Similarly, we demonstrated for the first time that a small molecule PDK-1 inhibitor, BX795, blocks HSV-1 protein synthesis. Both candidates were tested in vivo for topical treatment of corneal HSV-1 infection and we reported significant improvements in disease prognosis. The major goals for the next funding period are to: (1) improve the in vivo efficacy of DApt, (2) understand the molecular basis of antiviral action by BX795, and (3) develop BX795 for systemic treatment of ocular herpes. In the first specific aim, we propose several ways to improve efficacy. We will engineer therapeutic contact lenses for sustained delivery of DApt, improve DApt formulation to include permeating agents such as cylodextrins to increase the depth to which the aptamer is delivered into the corneal tissue, and develop higher efficacy (synergistic or additive) drug combinations using DApt and BX795 or a nucleoside analog. In the second specific aim, we will decode the molecular mechanisms responsible for the antiviral activity of BX795 using both biased and unbiased approaches. Focus on the cap- dependent translation pathways responsible for viral protein synthesis will constitute the biased approach; cell- free and whole cell proteomic analyses using quantitative mass spectrometry and study of post translational modifications will constitute unbiased approaches. In the third specific aim, we will comprehensively study the pharmacokinetic and toxicological profiles of intravenously administered BX795. Using this data, we will finally understand the safe and effective dosage of BX795 required for the effective inhibition of primary and reactivated HSV-1 infection of the cornea using murine models of corneal infection. Taken together our studies will establish novel antiviral mechanisms and help design new prophylactic and therapeutic ways to control HSV-1 infection of the eye.